Shan-Chwen Chang

A Novel Virulence Gene in Klebsiella pneumoniae Strains Causing Primary Liver Abscess and Septic Metastatic Complications

Primary Klebsiella pneumoniae liver abscess complicated with metastatic meningitis or endophthalmitis is a globally emerging infectious disease. Its pathogenic mechanism remains unclear. The bacterial virulence factors were explored by comparing clinical isolates. Differences in mucoviscosity were observed between strains that caused primary liver abscess (invasive) and those that did not (noninvasive). Hypermucoviscosity correlated with a high serum resistance and was more prevalent in invasive strains (52/53 vs. 9/52; P < 0.0001). Transposon mutagenesis identified candidate virulence genes. A novel 1.2-kb locus, magA, which encoded a 43-kD outer membrane protein, was significantly more prevalent in invasive strains (52/53 vs. 14/52; P < 0.0001). The wild-type strain produced a mucoviscous exopolysaccharide web, actively proliferated in nonimmune human serum, resisted phagocytosis, and caused liver microabscess and meningitis in mice. However, magA − mutants lost the exopolysaccharide web and became extremely serum sensitive, phagocytosis susceptible, and avirulent to mice. Virulence was restored by complementation using a magA-containing plasmid. We conclude that magA fits molecular Kochs postulates as a virulence gene. Thus, this locus can be used as a marker for the rapid diagnosis and for tracing the source of this emerging infectious disease.

Klebsiella pneumoniae Genotype K1: An Emerging Pathogen That Causes Septic Ocular or Central Nervous System Complications from Pyogenic Liver Abscess

BACKGROUND Since 1986, researchers have noted a syndrome of Klebsiella pneumoniae pyogenic liver abscess that is complicated by endophthalmitis or central nervous system infections. There are limited data regarding the role of bacterial genotype in the pathogenesis of this syndrome. METHODS We conducted a retrospective cohort study involving 177 cases of K. pneumoniae pyogenic liver abscess treated during 1997-2005 at a tertiary university hospital in Taiwan. We performed bacterial cps genotyping by polymerase chain reaction detection of serotype-specific alleles at wzy and wzx loci and used an in vitro serum assay to evaluate the virulence of bacterial strains. RESULTS Septic ocular or central nervous system complications developed in 23 patients (13%). Logistic regression analysis showed that genotype K1 was the only significant risk factor (adjusted odds ratio, 4.8; 95% confidence interval, 1.5-15.7, P=.009). The serum resistance assay indicated that, on average, K1 strains (n=100) were significantly more virulent than were strains of K2 (n=36), K20/K5/K54 (n=21), or other genotypes (n=20) (P<.001 for each comparison). In addition to the serotype-specific cps region, the genomic background of K1 strains also differed significantly from that of non-K1 strains (20-kb kfu/PTS region, 97/100 vs. 13/77; P<.001). Of the 19 cases in which genotype K1 strains caused complications, 8 patients (42%) did not have identifiable underlying medical diseases. CONCLUSIONS K. pneumoniae genotype K1 is an emerging pathogen capable of causing catastrophic septic ocular or central nervous system complications from pyogenic liver abscess independent of underlying diseases in the host.

Genetic Determinants of Capsular Serotype K1 of Klebsiella pneumoniae Causing Primary Pyogenic Liver Abscess

BACKGROUND Primary pyogenic liver abscess (PLA) caused by Klebsiella pneumoniae is an emerging infectious disease. Capsular serotype K1 and the magA gene have been reported to be associated with this disease. METHODS The prevalence of magA was determined by polymerase chain reaction (PCR). The sequences of the magA flanking region were completed by inverse PCR and direct sequencing. Serotyping was performed by double immunodiffusion. Insertion mutations and trans-complementation were used to define the K1 genetic determination region. RESULTS Thirty-five of 42 strains from patients with PLA were magA positive, whereas only 1 of 32 non-PLA strains was magA positive. All 36 magA-positive strains were serotype K1, and the 38 magA-negative strains were not (36/36 vs. 0/38; P<.0001). Sequencing of the magA flanking region revealed a putative capsular polysaccharide synthesis (cps) region; this region was 25 kb in length and contained 20 open reading frames (ORFs); of these ORFs, 9 were cotranscribed as part of an operon and differed from both MGH78578 and the Chedid strain. Mutation of 4 genes in this region turned the mutant strains anti-K1 negative. Trans-complementation restored the K1 phenotype. CONCLUSIONS The operon containing magA is responsible for capsular serotype K1 of K. pneumoniae. Several loci in the operon are unique determinants of K1 strains.

Antimicrobial drug resistance in pathogens causing nosocomial infections at a university hospital in Taiwan, 1981-1999.

To determine the distribution and antimicrobial drug resistance in bacterial pathogens causing nosocomial infections, surveillance data on nosocomial infections documented from 1981 to 1999 at National Taiwan University Hospital were analyzed. During this period, 35,580 bacterial pathogens causing nosocomial infections were identified. Candida species increased considerably, ranking first by 1999 in the incidence of pathogens causing all nosocomial infections, followed by Staphylococcus aureus and Pseudomonas aeruginosa. Candida species also increased in importance as bloodstream infection isolates, from 1.0% in 1981-1986 to 16.2% in 1999. The most frequent isolates from urinary tract infections were Candida species (23.6%), followed by Escherichia coli (18.6%) and P. aeruginosa (11.0%). P. aeruginosa remained the most frequent isolates for respiratory tract and surgical site infections in the past 13 years. A remarkable increase in incidence was found in methicillin-resistant S. aureus (from 4.3% in 1981-1986 to 58.9% in 1993-1998), cefotaxime-resistant E. coli (from 0% in 1981-1986 to 6.1% in 1993-1998), and cefotaxime-resistant Klebsiella pneumoniae (from 4.0% in 1981-1986 to 25.8% in 1993-1998). Etiologic shifts in nosocomial infections and an upsurge of antimicrobial resistance among these pathogens, particularly those isolated from intensive care units, are impressive and alarming.

Comparison of Both Clinical Features and Mortality Risk Associated with Bacteremia due to Community-Acquired Methicillin-Resistant Staphylococcus aureus and Methicillin-Susceptible S. aureus

BACKGROUND The majority of research about community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection has focused on skin and soft-tissue infections. No literature has been published on the clinical features and outcomes of adult patients with CA-MRSA bacteremia in comparison with patients with community-acquired methicillin-susceptible S. aureus (CA-MSSA) bacteremia. METHODS From 1 January 2001 through 31 December 2006, the demographic data and outcome of 215 consecutive adult patients admitted to a tertiary care center in Taiwan with S. aureus bacteremia (age, >16 years) who fulfilled the criteria for community-acquired S. aureus bacteremia were collected for analysis. RESULTS The mean age (+/-SD) was 56.8+/-20.5 years. There were 30 patients (14%) with CA-MRSA bacteremia and 185 (86%) patients with CA-MSSA bacteremia. Cutaneous abscess (odds ratio, 5.46; 95% confidence interval, 1.66-17.94) and necrotizing pneumonia (odds ratio, 24.81; 95% confidence interval, 2.63-234.03) were the independent predictors of CA-MRSA bacteremia; endovascular infection was the only independent predictor of CA-MSSA bacteremia. After Cox regression analysis, the independent significant risk factors for 30-day mortality included increased age, shock, and thrombocytopenia (<100,000 cells/microL). After adjustment, the day 30 mortality of patients with CA-MRSA bacteremia was not significantly higher than that of patients with CA-MSSA bacteremia (adjusted hazard ratio, 1.01; 95% confidence interval, 0.30-3.39; P = .986). Most (92%) of 25 available CA-MRSA isolates were multilocus sequence typing 59. CONCLUSIONS The number of adult patients with CA-MRSA bacteremia increased with time, and the disease was associated with more necrotizing pneumonia and cutaneous abscess but less endovascular infection than was CA-MSSA bacteremia. Patients with CA-MRSA bacteremia did not have higher mortality than did patients with CA-MSSA, even though most of the patients with CA-MRSA bacteremia did not receive empirical glycopeptide therapy.

Candida guilliermondii, an Opportunistic Fungal Pathogen with Decreased Susceptibility to Fluconazole: Geographic and Temporal Trends from the ARTEMIS DISK Antifungal Surveillance Program

ABSTRACT Although a rare cause of invasive candidiasis, Candida guilliermondii has been reported to exhibit decreased susceptibility to antifungal agents. Aside from case reports and small surveys, there is little information regarding the epidemiology and antifungal susceptibility profile of C. guilliermondii. We report geographic and temporal trends in the isolation and antifungal susceptibilities of 1,029 C. guilliermondii clinical isolates collected from 127 medical centers as part of the ARTEMIS DISK Antifungal Surveillance Program. In addition, we report the in vitro susceptibility of 132 bloodstream isolates of C. guilliermondii to caspofungin. C. guilliermondii represented 1.4% of the 75,761 isolates collected from 2001 to 2003 and was most common among isolates from Latin America (3.7% versus 0.6 to 1.1%). Decreased susceptibility to fluconazole was noted (75% susceptible; range, 68 to 77% across regions), and voriconazole was more active in vitro against C. guilliermondii than fluconazole (91% susceptible; range, 88 to 93% across regions). Fluconazole was least active against isolates from dermatology (58%) and surgical (69%) services and against isolates associated with skin and soft tissue infection (68%, compared to 85% susceptible for bloodstream isolates). There was no evidence of increasing azole resistance over time among C. guilliermondii isolates tested from 2001 to 2003. Of 132 bloodstream isolates of C. guilliermondii tested against caspofungin, most were inhibited by ≤2 μg/ml (96%; MIC50/MIC90, 0.5/1.0 μg/ml). C. guilliermondii, a species that exhibits reduced susceptibility to fluconazole, is the sixth most frequently isolated Candida species from this large survey and may be an emerging pathogen in Latin America.

Influence of Genospecies of Acinetobacter baumannii Complex on Clinical Outcomes of Patients with Acinetobacter Bacteremia

BACKGROUND acinetobacter baumannii complex infections are increasing in prevalence and are associated with a high mortality. Biochemical classification tests cannot differentiate A. baumannii (genospecies 2) from other genospecies. Genospecies typing offers a potential tool to determine whether there are major differences in pathogenicity among the genospecies. METHODS adult patients with A. baumannii complex bacteremia in intensive care units were prospectively observed from January 2007 through July 2009. A. baumannii complex was identified by biochemical methods and the Phoenix bacterial identification system. Genospecies were identified by 16S-23S ribosomal RNA intergenic-spacer sequencing. RESULTS among the 135 patients with A. baumannii complex bacteremia, 87 (64.4%) had isolates that belonged to genospecies 2, 36 (26.7%) had isolates that belonged to genospecies 13TU, and 12 (8.9%) had isolates that belonged to genospecies 3. Patients with A. baumannii (genospecies 2) bacteremia were more likely to have pneumonia than were patients with bacteremia due to genospecies 13TU (63.2 % vs 27.8%; P =.001), whereas patients with bacteremia due to genospecies 13TU were more likely to have primary bacteremia (69.4% vs 20.7%; P <.001). Genospecies 2 was less susceptible to antibiotics than were other genospecies. It was associated with a higher rate of mortality than was genospecies 13TU (58.6% vs 16.7%; P < .001). On multivariate analysis, genospecies 2 was an independent predictor of mortality (odds ratio, 5.46; 95% confidence interval, 2.00-14.91; P = .001). CONCLUSIONS genospecies 2 of the A. baumannii complex was associated with greater resistance to antibiotics and higher mortality among bacteremic patients, compared with other genospecies, especially genospecies 13TU. These findings emphasize the need to focus on genospecies to better understand the pathogenesis and epidemiology of infections caused by the A. baumannii complex.

Long-Acting Neuraminidase Inhibitor Laninamivir Octanoate versus Oseltamivir for Treatment of Influenza: A Double-Blind, Randomized, Noninferiority Clinical Trial

BACKGROUND A single administration of laninamivir octanoate, a long-acting neuraminidase inhibitor, against influenza infection has been proven effective in nonclinical studies. This study evaluated the clinical efficacy of laninamivir octanoate for the treatment of adult influenza patients. METHODS A double-blind, randomized controlled trial examined whether laninamivir octanoate was noninferior to oseltamivir. A total of 1003 patients aged ≥ 20 years with febrile influenza symptoms for no more than 36 h were randomized to receive either 40 mg of laninamivir octanoate, 20 mg of laninamivir octanoate, or oseltamivir. Laninamivir octanoate was inhaled once on day 1, and oseltamivir (75 mg) was administered orally twice daily for 5 days. The primary end point was the time to illness alleviation. RESULTS A total of 996 patients were included in the primary analysis (40-mg laninamivir octanoate, n = 334; 20-mg laninamivir octanoate, n = 326; and oseltamivir, n = 336). The median time to illness alleviation in the 40-mg laninamivir octanoate, 20-mg laninamivir octanoate, and oseltamivir groups was 73.0, 85.8, and 73.6 h, respectively. The difference between laninamivir octanoate and oseltamivir was -0.6 h (95% confidence interval, -9.9 to 6.9 h) for the 40-mg group and 12.2 h (95% confidence interval, -1.5 to 17.2 h) for the 20-mg group. The upper limits of the 95% confidence intervals were less than the prespecified noninferiority margin (18 h). The proportion of patients shedding virus at day 3 was significantly lower in the 40-mg laninamivir octanoate group than in the oseltamivir group (P = .006). CONCLUSIONS A single inhalation of laninamivir octanoate is effective for the treatment of seasonal influenza, including that caused by oseltamivir-resistant virus, in adults. CLINICAL TRIALS REGISTRATION NCT00803595.

Dengue Type 3 Virus in Plasma Is a Population of Closely Related Genomes: Quasispecies

ABSTRACT Using reverse transcription-PCR and clonal sequencing of the dengue virus envelope gene derived from the plasma samples of six patients, we reported for the first time that dengue virus circulates as a population of closely related genomes. The extent of sequence diversity varied among patients, with the mean pairwise proportions of difference ranging from 0.21 to 1.67%. Genome-defective viruses were found in 5.8% of the total number of clones analyzed. Our findings on the quasispecies nature of dengue virus and the defective virus in vivo have implications with regard to the pathogenesis of dengue virus.

Isolation of a Chromosomal Region of Klebsiella pneumoniae Associated with Allantoin Metabolism and Liver Infection

ABSTRACT Klebsiella pneumoniae liver abscess with metastatic complications is an emerging infectious disease in Taiwan. To identify genes associated with liver infection, we used a DNA microarray to compare the transcriptional profiles of three strains causing liver abscess and three strains not associated with liver infection. There were 13 clones that showed higher RNA expression levels in the three liver infection strains, and 3 of these 13 clones contained a region that was absent in MGH 78578. Sequencing of the clones revealed the replacement of 149 bp of MGH 78578 with a 21,745-bp fragment in a liver infection strain, NTUH-K2044. This 21,745-bp fragment contained 19 open reading frames, 14 of which were proven to be associated with allantoin metabolism. The K2044 (ΔallS) mutant showed a significant decrease of virulence in intragastric inoculation of BALB/c mice, and the prevalence of this chromosomal region was significantly higher in strains associated with liver abscess than in those that were not (19 or 32 versus 2 of 94; P = 0.0001 [χ2 test]). Therefore, the 22-kb region may play a role in K. pneumoniae liver infection and serve as a marker for rapid identification.